The present invention, in some embodiments thereof, relates to CHO cell lines, methods of generating same and uses thereof.
Recombinant therapeutic antibodies play an important role in treatment of a large variety of diseases. It is estimated that about 30% of the new coming drugs are likely to be based on antibodies in the next decade. Thirty recombinant antibodies and Fc fusion were approved for marketing with sales in 2008 that reached $35 billion.
Antibodies contain a target antigen-specific region which is composed of the variable regions of both the heavy and the light chains. This part of the antibody may bind and neutralize a soluble antigen or a membrane bound target.
The Fc portion is responsible for effector functions through antibody dependant cellular cytotoxicity (ADCC) mechanism, complement dependant complex (CDC) and the neonatal receptor FcRn. Those effector functions are mediated through interaction of the effector molecules with the hinge and CH2 regions of the Fc. The CH2 domain contains an oligosaccharide located on the N glycosylation site at position 297 of the antibody which is known to play an important role in binding to effector cells. The oligosaccharide is usually composed of a complex diantennary type with considerable heterogeneity, such as a core heptasaccharide together with additional variable outer sugar residues.
ADCC is one of the critical killing mechanisms for antibodies that bind ligands on target cells' membrane. FcγR expressed on leucocytes bind the CH2 domain of the antibodies and upon binding and creation of immune complexes with antigens on the target cells activation of the leucocytes is initiated. The activation may include phagocytosis and release of cell mediators that lead to cell permeabilization and death. The ADCC activity is dependent on the IgG isotype on the one hand, and on a specific FcγR, on the other hand. Whereas IgG1 and IgG3 may induce this activity, IgG4 does not. The FcγR that binds the IgG and is important for ADCC mechanism activation is known as the FcγRIIIa and is expressed on NK cells and macprophages. In many cases the ADCC activity obtained upon binding of the NK cell to the target cell is not efficient enough to perform killing of the target cell. The reason is that the affinity of the FcγRIIIa to the IgGI is low.
Increased ADCC activity was found in patients expressing the high affinity allotype FcγRIIIa—158Val found in 10-15% of the population in comparison to patients expressing the FcγRIIIa—158Phe. Elevated ADCC was obtained also by manipulations done on the IgG Fc. Computational design algorithms were used in order to engineer antibodies and select for high affinity by high-throughput screening. This work yielded antibodies which display >2 orders of magnitude enhancement of in vitro effector function (Lazar, Dang et al. 2006), although decrease thermostability of mutated IgG1 (IgG1 with mutations S239D, A330L and I332E) was detected. Another approach for obtaining antibodies with enhanced ADCC is to produce them with low fucose levels on their oligosaccharide at position 297. Previously it was found that fucose residues on the oligosaccharide interfere with Fc binding to FcγRIIIa (Shinkawa, Nakamura et al. 2003). One way for obtaining antibodies with low fucose levels is to harness cells with such natural capabilities, such as Rat hybridoma YB2/0 cells (Shinkawa, Nakamura et al. 2003), although recombinant proteins produced in these cells have variable levels of fucose content. Several other possibilities of non-mammalian cells include avian cells from Vivalis, engineered aquatic plant Lemna from Biolex (Cox, Sterling et al. 2006) and variant of the moss Physocmirtella patens from Igeneon (Nechansky, Schuster et al. 2007). In addition, GlycoFi generated various lines of Pichia Pastoris cells with capabilities for several glycosylation solutions including enhanced ADCC (Hamilton, Davidson et al. 2006). Also several mammalian cells are used for production of antibodies with various glycosylation solutions in general and enhanced ADCC in particular. Glycotope created various human glycoengineered cell lines to glyco-optimize bio-therapeutics glycosylation. Glycart, acquired by Roche, engineered a cell line producing recombinant antibodies with reduced fucose level by introducing beta(1,4)—N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC) (Umana, Jean-Mairet et al. 1999). Biowa generated a knockout in the fucosyl transference 8 (Fut8) gene of CHO DG44 in order to diminish the fucose levels (Yamane-Ohnuki, Kinoshita et al. 2004).
A recent research demonstrated that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-D-lyxo-4-hexylose reductase within the cytosol can block the conversion of the intermediate GDP-4-keto-6-deoxymannose into a dead-end product that typically does not occur in vertebrate cells. Therefore CHO cells that were modified in this way secreted antibodies lacking core fucose (von Horsten, Ogorek et al.). Another approach was to create lectin resistant mutants that survive in the presence of toxic fucose specific lectin. LEC13 is a CHO based cell line that was developed by incubation of CHO cells in the presence of toxic pea fucose specific lectin (Ripka and Stanley 1986). LEC13 is deficient in GDP-mannose 4,6-dehydratase activity which results in expression of human IgG1 that are deficient of fucose (Shields, Lai et al. 2002).
U.S. Patent Application No. 2010/0081150 teaches mutation of CHO cells by treatment with chemicals and selecting cells which exhibit a variant glycosylation pattern including a decrease in fucosylation by killing cells with high fucose, in order to generate cells useful for expressing antibodies.
U.S. Patent Application No. 2010/0304436 teaches fucosylation reduced CHO cell lines by mutation in the Fx protein and controlling the availability of an external source of fucose in order to direct the ability of the cells to fucosylate polypeptides.
Kanda et al, (Kanda, Imai-Nishiya et al. 2007) discloses GMD and FUT8 knockout host cell lines. The GMD knockout cells have a genomic deletion corresponding to GMD exon 5, 6 and 7 regions.
Ripka et al. (Ripka and Stanley 1986) discloses four lectin resistant CHO mutant cells. The mutations were effected by incubation with N-methyl-N-nitrosoguanidine.
Shields et al, (Shields, Lai et al. 2002) discloses that IgG1 produced by the Lec13 (fucose deficient CHO) cell line increased binding to FcγRIIIA up to 50 fold and also increased ADCC.
Kanda et al., (Kanda, Yamane-Ohnuki et al. 2006) discloses that Lec13 produces 50-70% fucosylated antibody; however this known cell line could not stably produce antibody. Thus the Lec13 cell line is not suitable as a production cell line.